The detection and diagnosis of disease is of obvious importance for the treatment of disease. Numerous characteristics of diseases have been identified and many are used for the diagnosis and prognosis of disease. Many diseases are preceded by, and are characterized by, changes in the state of the affected cells. Changes may include the expression of viral genes in infected cells, changes in the expression patterns of genes in affected cells, changes in enzymatic activities, and changes in cell morphology. The detection, diagnosis, and monitoring of diseases may be aided by the assessment of such cell states, especially by improving the accuracy of detection.
The worldwide annual incidence of cervical cancer is approximately 500,000 cases1,2. The current diagnostic assay for early cervical cancer detection is the Papanicolaou (Pap) test which is a simple morphological screening method of examining strained exfoliative cells. It is used most commonly to detect cancers of the cervix, but it may be used for tissue specimens from any organ. The findings are usually reported descriptively and grouped into several classifications, including the Papanicolaou and the cervical intraepithelial neoplasia (CIN) classifications.
The Pap test contributed to a 74% decline in deaths due to cervical cancer in the United States between 1955 and 19923. However, the sensitivity of the Pap test for high-grade cervical lesions (cervical intraepithelial neoplasia grade 3 or CIN 3) is not very high, typically ranging from as low as 50% for conventional Pap tests to 85% for the newer liquid cytology tests4. Routine use of advanced cytology-based automated reading technologies such as Auto-Pap® 300 QC, AUTOCyt®, and Cyto-Savant™ is not a favorable approach due to high cost5 and only minor increases in sensitivity for CIN 36. The clinical data suggest that HPV DNA tests are more sensitive (about 90 to 95%) than either the conventional (50 to 70%) or liquid-based (60 to 85%) Pap tests. Combination of the Pap test with the HPV DNA test reduces false-negative cases and produces combined sensitivity for CIN 3 at 95% to 100%. However, false negative cytology tests account for at least 25% of invasive cancer cases in the United States7. Additionally, false-positive cases are another important limitation of the cytology approach.
Alternative diagnostic approaches are to couple the Pap test with a test for either HPV DNA8,9,10 or cellular molecular markers11,12,13. The combination of the Pap test with a HPV DNA test achieves high sensitivity and can partially address the relatively high rate of HPV positive cases in women without prevalent cervical neoplastic disease. There is an increasing acceptance for those who apply appropriate algorithms in recommending women who are positive for HPV alone to make follow up appointments with longer intervals compared to those women who are positive for both the HPV nucleic acid test and the Pap test who are recommended to have immediate attention. The use of these algorithms and recommendations will be of clinical benefit. High-risk HPV DNA is highly associated with cervical cancer14, however, most HPV infections do not lead to cervical cancer. It is estimated that 5% to 10% of normal women are HPV infected by carcinogenic HPV types, and of these up to one-quarter may be expected to develop high grade cervical lesions15. Women over 30 years of age are more likely to develop neoplastic disease than younger women16.
Two formal classification systems are utilized for identification of cervical cancer precursor conditions. The CIN system relates to the tissue biopsies classification and comprises of Negative; mild cervical dysplasia or CIN 1; moderate dysplasia or CIN 2; severe dysplasia (including carcinoma in situ) or CIN 3; and carcinoma. The Bethesda Classification system relates to Pap changes and comprises of within normal limits (WNL) and benign cellular changes (equivalent to Negative); atypical squamous cell of undetermined significance (ASCUS) or atypical glandular cell of undetermined significance (AGCUS) favor benign (no equivalent in the CIN system); ASCUS or AGCUS favor dysplasia (no equivalent in the CIN system); low grade squamous intraepithelial lesion (LSIL) (equivalent to CIN 1); high grade squamous intraepithelial lesion (HSIL) (equivalent to CIN 2-3); and carcinoma. (PATH Outlook Vol 18, # 1, 2000; Jelovsek FR Woman's Diagnostic Cyber).
Human papillomavirus (HPV) induces benign epithelial proliferations of the skin and mucosa in humans and is associated with anogenital neoplasias and carcinomas. Human papillomaviruses characterized to date are associated with lesions confined to the epithelial layers of skin, or oral, pharyngeal, respiratory, and, most importantly, anogenital mucosae. HPV is a virus that is the cause of common warts of the hands and feet, as well as lesions of the mucous membranes of the oral, anal, and genital cavities. The virus may be transmitted through sexual contact and is a precursor to cancer of the cervix. Non-limiting examples of diseases, disorders, and conditions associated with HPV include cervical intraepithelial neoplasia (CIN), and cancer, cervical dysplasia, vaginal cancer, vaginal dysplasia, vulvar cancer, penile cancer, anal cancer, oral cancer, atypical squamous cells including atypical squamous cells of undetermined significance (ASCUS) and atypical squamous cells, high-grade squamous intraepithelial lesion (HSIL), genital warts, plantar warts, butcher's warts, and flat warts, condylomata, epidermo dysplasia verruciformis and other skin diseases, laryngeal papilloma, oral papilloma and conjunctival papilloma. More than 70 types of HPV have been identified, many of which have been isolated from anogenital lesions.
HPV types may be divided into three groups according to their biological oncogenic potential, where some are associated with cancerous and pre-cancerous conditions. Low-risk HPV types are more frequently associated with low grade squamous intraepithelial lesions (LSIL; suspect CIN 1 lesions) and condyloma acuminatum. This group comprises, but is not limited to, low-risk HPV types 6, 11, 42, 43, 44, and others. Intermediate-risk HPV types comprise, but are not limited to HPV types 31, 33, 35, 39, 51, 52, 56, 58, 59, and 68. High-risk HPV types comprise, but are not limited to HPV types 16, 18, and 45. High-risk HPV types are more frequently associated with high-grade squamous intraepithelial lesions (HSIL; suspect CIN 2, CIN 3) and invasive carcinoma of uterine cervix.
The viral genome may be divided into three regions: (1) the upstream regulatory region (URR) or long control region (LCR), containing control sequences for HPV replication and gene expression; (2) the viral early gene region, encoding, among others, the E2, E6 and E7 genes; and (3) the late region, encoding the L1 and L2 genes.
HPV gene expression in high-grade premalignant disease or cancer appears restricted to the early genes, possibly due to cellular differentiation arrest induced by the viral E6 and E7 genes. In comparison to active HPV infection, E6 and E7 gene control in cancer is deranged by mutations in the viral URR and, in integrated viral fragments, by the disruption of the viral E2 gene, stabilization of E6 and E7 mRNAs, and influences at the cellular integration site. Cervical cells containing extrachromosomal HPV genomes rapidly segregate and are outgrown in culture by cells that contain integrated viral genomes17.
Cervical neoplastic progression and cancer are highly associated with HPV persistence and viral DNA integration in the cellular genome. These primary molecular events, characteristic for high-risk HPV DNA subtypes, result in over-expression of the viral oncogenes E6 and E7. The presence of high-risk HPV E6 and E7 proteins leads to inactivation of the important tumor-suppression proteins p53 and pRB and their associated pathways18. As a result, p53- and pRB- associated genes and their products (mRNA and protein) are aberrantly expressed. Some aberrantly expressed gene products are called markers due to their readily detectability and association with neoplastic progressions19. Thus, another alternative approach to improve sensitivity and/or specificity of the Pap test is to combine with marker-based screening. However, the combination of Pap test with molecular markers will also have to account for false negatives and false positives due to limited marker sensitivity and specificity (without information on high-risk HPV DNA) for neoplastic progression13. The published studies show high sensitivity of the molecular markers for high grade squamous intraepithelial lesion (HSIL) specimens. However, a high rate of certain marker-positive cases (approximately 12%-26%) for the within normal limits (WNL) group, (approximately 74%-88%) for atypical squamous cells of undetermined significance (ASCUS) and low grade squamous intraepithelial lesion (LSIL) groups have also been demonstrated11,20,21,23. A high-rate of false positive cases is thus evident for some marker and Pap test combinations.
Several methods have been used to diagnose clinical or subclinical infection with HPVs including clinical observation, cytological screening by Pap test, electron microscopy, immunocytochemistry, and DNA hybridizations. However, a definitive diagnosis of HPV infection depends on the detection of nucleic acids (DNA or RNA) or proteins of the virus. The detection of HPV DNA by nucleic-acid-based assays may identify 20-30% of women with cervical disease who have false-negative results by Pap test screening. The presence of HPV DNA in the cervical lesions of older women has a higher predictive value for the progression of cervical intraepithelial neoplasia to invasive cancer than in younger women. Thus, there is a need for a more accurate, sensitive, and efficient method of screening clinical specimens for neoplastic disease than the time consuming and subjective Pap cytology test screenings. There is also a need for further improvements to HPV nucleic acid testing alone or the combination of Pap test screening and either HPV DNA or molecular marker tests.
Accordingly, it is an object of the present invention to provide an accurate, sensitive, and efficient method for molecular diagnosis and prognosis of HPV-based disease, where the method as a whole incorporates added specificity, enabling individual steps of the method to use lower stringency.
It is another object of the present invention to provide an assay to improve the accuracy and reliability of diagnostic and prognostic assessments of HPV-based disease.
It is a further object of the present invention to provide a method for assessing the risk that a patient infected with HPV will have or will develop HPV-based high grade neoplastic disease.
Yet a further object of the present invention is to provide a method for monitoring the effectiveness of treatment of HPV-based disease.
Another object of the present invention is to provide a scale identifying the degree of HPV high risk disease progression.
A further object of the present invention to provide kits for diagnosing, prognosing, and assessing the stage of HPV-based disease.